The Mechanism Of Beta-globin Gene Silencing In Embryonic

已在K562细胞中研究了支配人β-珠蛋白基因转录的发育特异性的分子机制，K562细胞是表达最小β-珠蛋白的人红白血病细胞系。蛋白质结合分析显示5'区含有三个由反式作用因子结合的元件，β-蛋白1（BP 1）和β-蛋白2（BP 2）。在含有氯霉素乙酰转移酶（pCAT）的β-珠蛋白载体中的每个元件的体外诱变，随后瞬时转染到K562细胞中，使CAT活性水平比野生型β-CAT高5.5倍，这与它们的沉默作用一致。BP 1和BP 2基序具有与高迁移率族蛋白（HMG 1 +2）重叠的结合位点，通过环状排列分析显示DNA弯曲因子使β-珠蛋白远端启动子外切弯曲。理论上，所有β-蛋白结合位点的突变都可能影响HMG 1 +2的结合，足以阻碍促进低水平组成型基因表达所需的DNA-蛋白和/或蛋白-蛋白相互作用。在BP 1和BP 2基序之间放置两个螺旋圈的DNA也增加了3倍的表达，这表明了最佳沉默所需的空间限制。然而，插入HMG 1 +2 DNA弯曲基序（也相当于两圈）通过重建BP 1-BP 2邻近性促进β沉默。因此，一般的DNA弯曲和特定的转录因子的组合似乎参与β-珠蛋白沉默在胚胎/胎儿红系阶段。为了进一步确定这种关系，正在进行使用转基因动物的体内研究。我们已经启动了实验，以建立一个转基因小鼠模型，用于通过BP 1蛋白特异性抑制成人β-珠蛋白表达。我们在含有micro-LCR的35 kb粘粒构建体的β-珠蛋白基因序列的远端启动子中引入了突变的BP 1结合位点（基因座控制区）和人β-珠蛋白基因簇的所有其它必需元件。该构建体和适当的对照已经被显微注射到小鼠卵中。随后的转基因应允许在胚胎-胎儿发育阶段的BP 1沉默基序的作用在体内分析。

The molecular mechanisms which govern the developmental specificity of human beta-globin gene transcription has been studied in K562 cells, a human eyrthroleukemia line that expresses minimal beta-globin. Protein-binding analysis reveals the 5' region contains three elements bound by trans-acting factors, beta-protein 1 (BP1) and beta-protein 2 (BP2). In vitro mutagenesis of each element in a beta-globin vector containing chloramphenicol acetyltransferase (pCAT) followed by transient transfection into K562 cells increased levels of CAT activity 5.5-fold higher than wt beta-CAT, consistent with their silencing role. Mutagenesis of all three elements, however, resulted in activity significantly lower than wt beta-CAT. BP1 and BP2 motifs have overlapping binding sites with high mobility group proteins (HMG1+2), DNA-bending factors shown here by circular permutation assay to extrinsically bend the beta-globin distal promoter. Theoretically, mutations in all beta-protein binding sites could affect the binding of HMG1+2 sufficiently to impede DNA-protein and/or protein-protein interactions needed to facilitate the low level, constitutive gene expression. Placing two helical turns of DNA between BP1 and BP2 motifs also increased expression 3-fold, indicative of spatial constraints required for optimal silencing. However, insertion of the HMG1+2 DNA-bending motif (also equivalent to two turns) facilitates beta-silencing by re-establishment of BP1-BP2 proximity. Thus a combination of general DNA-bending and specific transcriptional factors appear to be involved in beta-globin silencing in the embryonic/fetal erythroid stage. To further define this relationship an in vivo study using transgenic animals is underway. We have initiated experiments to establish a transgenic mouse model for specific repression of adult beta-globin expression by the BP1 protein.To alter the developmental specificity of the human beta-globin gene, we introduced a mutated BP1 binding site into the distal promoter of beta-globin gene sequence of 35 kb cosmid constuct containing the micro-LCR (locus control region) and all other essential elements of the human beta-globin gene cluster. This construct and appropriate controls have been mico-injected into mouse eggs. The subsequent transgeneics should permit the in vivo analysis of the role of BP1 silencing motif during the embryonic-fetal developmental stages.